The present invention relates to methods of production of the completely post-translationally modified protein by combination of cell-free protein synthesis and cell-free complete post-translational modification.
The present invention relates to protein production of therapeutic, research and commercial value requiring post-translational modification not by a cell culture method but a cell-free completely post-translationally modified protein synthesis method. To explain in more detail, using a cell-free completely post-translationally modified protein synthesis method comprised of cell extract including protein synthesis machinery and cell membrane extract including complete post-translational modification machinery, a valuable protein is produced.
Many pharmacological proteins undergo post-translational modifications such as glycosylation, phosphorylation and amidation etc. that are indispensable for their activity. Moreover, the majority of proteins secreted by mammalian cells are post-translationally modified. Many proteins become post-translationally modified during the xe2x80x98secretory processxe2x80x99, which comprises of a journey from their site of synthesis in the rough endoplasmic reticulum (ER), through the Golgi apparatus and then to various cellular or extracellular destinations. In post-translational modification of a protein, inorganic phosphate is attached to proteins through phosphorylation, amide group is generated at the terminus of polypeptides by amidation and carbohydrate is attached to proteins through glycosylation. Of these, the most complex procedure involving several enzymes is glycosylation. Hence, glycoproteins are the most diverse group of biological compounds that are ubiquitous constituents of almost all living organisms. They occur in cells in both soluble and membrane-bound forms as well as in the extracellular matrix and in intercellular fluids, and they serve a variety of functions. These proteins possess oligosaccharides covalently attached through an asparagine (Asn) side chain (xe2x80x9casparagine-linkedxe2x80x9d or xe2x80x9cN-linkedxe2x80x9d) or through a threonine or serine side chain (xe2x80x9cO-linkedxe2x80x9d). A given glycoprotein may contain N-linked oligosaccharide chains only, O-linked oligosaccharide chains only, or both. The carbohydrate units of glycoproteins exhibit considerable variation in size and structure ranging from mono- or disaccharide to a branched oligosaccharide composed of as many as 20 monosaccharide residues.
In summary, N-linked glycosylation begins with the synthesis of a lipid-linked oligosaccharide moiety, and its transfer en bloc to a nascent polypeptide chain in the ER. Attachment occurs through Asn, generally at the tripeptide recognition sequence Asn-X-Ser/The, where X can be any amino acid from naturally occurring amino acids. A series of trimming reaction is catalyzed by exoglycosidase in the ER. Further processing of N-linked oligosaccharides by mammalian cells continues in the compartment of the Golgi, where a sequence of exoglycosidase- and glycosyltransferase-catalyzed reactions generate high-mannose, hybrid-type and complex-type oligosaccharide structures.
For the biosynthesis of post-translationally modified proteins with biological activity, it is necessary to culture eukaryotic cells capable of undergoing the post-translational modification including glycosylation. The production of post-translationally modified proteins by the culture of such cells, however, costs a great deal, e.g., in right of culture medium cost, operation cost, apparatus cost, etc. This process is also very time-consuming and in order to simplify the process and to save time, many cell-free protein synthesis systems have been developed.
A cell-free protein synthesis has been used as an experimental tool for the investigation of gene expression in vitro especially for the proteins that cannot be synthesized in vivo because of their toxicity to host cells. In addition, various synthetic amino acids besides the 20 natural amino acids can be effectively introduced by this method into protein structures for specially designed purposes (Noren, C. J. et al., Science 244:182-188 (1989)). Moreover, the cell-free protein synthesis has been recently re-evaluated as an alternative to the production of commercially important recombinant proteins, which is mainly due to the recent development of a novel reactor system and the extensive optimization of reactor operating conditions (Kim, D. M. et al., Eur. J. Biochem. 239:881-886 (1996); Kigawa, T. et al., FEBS Lett. 442:15-19 (1999)). Therefore, the development of cell-free protein synthesis systems has reached the next stage, i.e., the production of active proteins on a commercial scale.
Accordingly, some methods were developed to produce the co-translationally modified and the initial post-translationally modified proteins by adding organelles relevant to the co-translational and the initial post-translational modification to these cell-free protein synthesis systems. As a representative method, U.S. Pat. No. 6,103,489 discloses that cell-free assay systems for proteins with the co-translational and the initial post-translational modification have been constructed by combining a eukaryotic cell-free translation system with rough microsome. In another method, the extract including translational components and post-translational modification components such as ER was prepared by a single step extraction from a single source (Hiroshi, T., et al., J. Biosci. Bioeng. 5:508-514 (2000)). In cells, the biosynthesis of many proteins requires co-translational translocation across membranes of an organelle called ER for proper processing. In cell-free systems, in place of the ER, microsomal membranes are used, which are equivalent to the ER in that they contain a high percentage of ER membrane which have been isolated by centrifugation. These reconstituted assay systems for assessing protein translation and the initial post-translational processing in higher eukaryotes have allowed characterization of the translocational machinery, and are being actively used to define the topology of membrane proteins and to elucidate the regulation of N-linked core glycosylation. However, they do not produce the completely post-translationally modified proteins. Since proteins not completely post-translationally modified do not have the complete and correct structure, they cannot be used to study the utility and characteristics of proteins including pharmacological activities. This is due to the fact that proteins with incomplete post-translational modification or no post-translational modification do not or have low biological activity and therefore cannot be used therapeutically. Current cell-free protein synthesis methods cannot produce proteins with a complete structure and therefore is insufficient to be used to study post-translational modifications or functions of genes.
In the meantime, from the results of the Human Genome Project, a great deal of information on the genetics of human have been obtained. In the near future, the complete genome map of humans being will be available. At present, we have reached the Post Genomic Era where the functions of genes and their encoded proteins are being studied. In order to achieve this goal, computer programs that can use information about the sequence of a gene to predict the structure of the encoded protein are used in bioinformatics. From the predicted structure of the protein and the sequence of the gene, the function of the protein can be elucidated. This process of obtaining the function of the protein through prediction is not a completely satisfactory method. A better method would be to express the protein and to study the function through experiments. The protein to be studied should have the complete co- and post-translational modifications. To produce these proteins, the genes of interest should be expressed in a eukaryotic host cell. This process is very time-consuming and in order to simplify the process and to save time, cell-free protein synthesis can be used. Up until now, a cell-free protein synthesis system with a complete post-translational modification has not been developed. Therefore, a cell-free protein synthesis system capable of producing a protein that has undergone complete post-translational modifications needs to be developed.
To solve the problem of prior art that cell-free protein synthesis systems produced incompletely post-translationally modified protein, the present invention provides methods of producing the completely post-translationally modified proteins by a more advanced cell-free protein synthesis system, in particular, by the combination of cell-free protein synthesis and cell-free complete post-translational modification.
We expect that the methods of cell-free protein synthesis according to the present invention can become a useful tool for synthesizing completely post-translationally modified protein for the functional analysis of proteins and the large-scale production of therapeutically important proteins, and to serve as a model system for elucidating the role of proteins and the mechanisms of post-translational modification.
In order to achieve such goal, the present invention provides a method for preparing completely post-translationally modified protein via coupled cell-free completely post-translationally modified protein synthesis comprising;
adding a DNA template to a cell extract,
adding ribonucleotide triphosphates to the extract, and
adding a sufficient amount of co- and post-translational modification machinery such as ER/Golgi apparatus, or ER/Golgi apparatus/plasma membrane, or other organelles in addition to these to the extract to stimulate the production of completely post-translationally modified protein,
or via uncoupled cell-free completely post-translationally modified protein synthesis comprising;
adding a RNA template to a cell extract, and
adding a sufficient amount of co- and post-translational modification machinery such as ER/Golgi apparatus, or ER/Golgi apparatus/plasma membrane, or other organelles in addition to these to the extract to stimulate the production of completely post-translationally modified protein.
The most complex post-translational modification process requiring several enzymes is glycosylation. Correct glycosylation implies that most post-translational modification is possible using the same method.
Reviewing the glycosylation process of proteins in cells, glycosylation in most eukaryotes occurs commonly in the ER, i.e., yeast, insect, plant and mammalian cells share the features of N-linked oligosaccharide processing in the ER. Though the resultant glycoproteins in the ER have a near identical carbohydrate structure, with only the initial glycosylation in the ER, glycoproteins with a therapeutic efficacy cannot be fully produced.
The production of premature glycoprotein, which does not undergo the complete post-translational modification, may be caused by the deficiency of the terminal glycosylation machinery such as the Golgi apparatus. In other words, oligosaccharide processing by different cell types may diverge in the Golgi apparatus. The initial step in O-glycosylation by mammalian cells is the covalent attachment of N-acetylgalactosamine to serine or threonine. No O-glycosylation sequence has been identified analogous to the Asn-X-Ser/Thr template required for N-glycosylation. In further contrast to N-glycosylation, no preformed, lipid-coupled oligosaccharide precursor is involved in the initiation of mammalian O-glycosylation. Sugar nucleotides serve as the substrates for the first and all subsequent steps in O-linked processing. Following the covalent attachment of N-acetylgalactosamine to serine or threonine, several different processing pathways are possible for mammalian O-linked oligosaccharides in the Golgi. The oligosaccharide structures of glycoproteins can have a profound effect on properties critical to the human therapeutic use, including plasma clearance rate, antigenicity, immunogenicity, specific activity, solubility, resistance to thermal inactivation, and resistance to protease attack. Therefore, for a cell-free protein synthesis to be applied to the large-scale production of glycoprotein and for a rapid insight into the role of protein glycosylation to understand the relationship among stability, conformation, function of protein and glycosylation, an efficient cell-free completely post-translationally modified protein synthesis system in which protein is completely post-translationally modified needs to be developed.
For the production of proteins having the complete and correct structure, the present invention includes the combination of a cell-free protein synthesis system and co- and post-translational modification machinery containing organelles, separated from cells, relevant to co- and post-translational modification. This cell-free completely post-translationally modified protein synthesis method is a new approach that has not been attempted by anyone. This method is suitable especially to large-scale production of efficacious and useful proteins. Additionally, this method can be applied directly to post-translational modification processes, required to produce a biologically active protein besides glycosylation.